[Skip to main content](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-254186230#main-content/index.html) Aflibercept as a VEGF Decoy Receptor Research reportView only Create alertChat May 5, 2026 # What is aflibercept's mechanism as a soluble decoy receptor that binds VEGF-A and PlGF? ## Aflibercept functions as a soluble decoy receptor by binding VEGF-A and PlGF with high affinity in 1:1 complexes, preventing these ligands from activating their native VEGFR1 and VEGFR2 receptors and thereby blocking downstream angiogenic signaling pathways. # Abstract Aflibercept functions as a soluble decoy receptor composed of the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2 fused to the Fc region of human IgG1. It binds all isoforms of human VEGF-A with subpicomolar affinity (Kd = 0.49-0.5 pM) and PlGF with low picomolar affinity (Kd = 38.9 pM for human PlGF-2), forming homogeneous 1:1 complexes with each VEGF dimer. This binding mechanism occludes both the amino acids necessary for VEGFR1/R2 binding and the heparin-binding site on VEGF165, preventing VEGF-A and PlGF from activating their native receptors. The association rate for aflibercept binding to VEGF-A is orders of magnitude faster than bevacizumab and ranibizumab, and unlike these antibody-based approaches, aflibercept also binds VEGF-B and PlGF, providing broader VEGF family inhibition. By sequestering VEGF-A and PlGF from circulation, aflibercept suppresses phosphorylation of VEGFR1 and VEGFR2 and downstream pathways including PI3K/AKT/mTOR and ERK, resulting in reduced endothelial cell proliferation, migration, and permeability. The monomeric 1:1 complex formation distinguishes aflibercept from bevacizumab’s multimeric complexes and avoids platelet activation and increased Fcγ receptor binding. An unexpected mechanistic property is aflibercept’s glycosylation-dependent binding to galectin-1, an angiogenic factor independent of VEGF-A, representing a novel anti-angiogenic function beyond VEGF family antagonism. Methods We analyzed 10 sources from an initial pool of 200, using 7 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods Records from Elicit search n = 200 Papers screened using: Aflibercept Mechanism Focus, Target Binding Interactions, Mechanistic Data Provision, Appropriate Methodology, Beyond Clinical Outcomes Only, Aflibercept Inclusion, Publication Type Adequacy n = 200 Papers screened out n = 190 Papers included for extraction n = 10 Press enter or space to select a node.You can then use the arrow keys to move the node around. Press delete to remove it and escape to cancel. Press enter or space to select an edge. You can then press delete to remove it or escape to cancel. ## Paper search We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of [Semantic Scholar](https://www.semanticscholar.org/) and [OpenAlex](https://openalex.org/). We ran this query: “What is aflibercept’s mechanism as a soluble decoy receptor that binds VEGF-A and PlGF?” The search returned 200 total results from Elicit. We retrieved 200 papers most relevant to the query for screening. ## Screening We screened in sources based on their abstracts that met these criteria: - **Aflibercept Mechanism Focus**: Does this study investigate aflibercept’s molecular mechanism of action as a decoy receptor? - **Target Binding Interactions**: Does this study examine aflibercept’s binding interactions with VEGF-A and/or PlGF? - **Mechanistic Data Provision**: Does this study provide mechanistic data through in vitro, in vivo, ex vivo, or clinical approaches? - **Appropriate Methodology**: Does this study use biochemical, biophysical, molecular biology, or structural techniques to characterize aflibercept? - **Beyond Clinical Outcomes Only**: Does this study provide mechanistic insights beyond solely reporting clinical efficacy or safety outcomes? - **Aflibercept Inclusion**: Does this study include aflibercept as a primary focus or in direct comparison (rather than investigating other anti-VEGF agents exclusively)? - **Publication Type Adequacy**: Is this study a full research article, review, or other substantial publication (not a case report, case series, conference abstract, or letter to editor without mechanistic investigation)? We considered all screening questions together and made a holistic judgement about whether to screen in each paper. ## Data extraction We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column. - **Binding Properties**: Extract all binding characteristics of aflibercept to VEGF-A and PlGF, including: - Binding affinity (Kd values, IC50, EC50) - Association and dissociation kinetics (ka, kd rates) - Binding stoichiometry (ratio of aflibercept to VEGF-A/PlGF) - Comparison of binding strength to VEGF-A vs PlGF - Any differences between VEGF-A isoforms (e.g., VEGF121, VEGF165) - Temperature, pH, or other conditions affecting binding - **Structural Mechanism**: Extract molecular details of how aflibercept binds VEGF-A and PlGF as a soluble decoy receptor, including: - Which domains of aflibercept (VEGFR1, VEGFR2, Fc) are involved in binding - Specific amino acid residues or regions important for binding - What sites on VEGF-A/PlGF are blocked by aflibercept binding - Whether heparin-binding sites are occluded - Formation of complexes (1:1, multimeric, etc.) - Any structural differences in binding to VEGF-A vs PlGF - **Decoy Receptor Function**: Extract evidence of how aflibercept functions as a decoy receptor to prevent normal VEGF-A and PlGF signaling, including: - Prevention of VEGF-A/PlGF binding to native VEGFR1 and VEGFR2 - Inhibition of receptor phosphorylation and activation - Sequestration/trapping of free VEGF-A and PlGF from circulation - Effects on ligand availability for endogenous receptors - Any evidence of competitive vs non-competitive inhibition mechanisms - **Downstream Signaling Effects**: Extract the signaling pathway consequences of aflibercept’s decoy receptor mechanism, including: - Effects on VEGFR1/VEGFR2 phosphorylation and activation - Impact on downstream pathways (PI3K/AKT, mTOR, MAPK, etc.) - Changes in cellular responses (proliferation, migration, permeability) - Effects on angiogenic processes (endothelial cell function, vessel formation) - Any feedback effects on VEGF/PlGF expression - Comparison of signaling blockade for VEGF-A vs PlGF pathways - **Ligand Specificity Profile**: Extract the complete ligand binding profile of aflibercept as a soluble decoy receptor, including: - All VEGF family members that aflibercept binds (VEGF-A, VEGF-B, PlGF, others) - VEGF family members that aflibercept does NOT bind - Relative binding strengths across different ligands - Species specificity (human vs mouse vs other species) - Any cross-reactivity with other growth factors or proteins - **Mechanistic Comparison**: Extract comparisons of aflibercept’s decoy receptor mechanism to other anti-VEGF agents, including: - Differences from antibody-based approaches (bevacizumab, ranibizumab) - Advantages/disadvantages of soluble receptor vs antibody mechanisms - Differences in complex formation (1:1 vs multimeric complexes) - Unique aspects of aflibercept’s binding compared to other agents - Any superior or inferior mechanistic properties noted - **Experimental Methods**: Extract the experimental approaches used to study aflibercept’s mechanism as a decoy receptor, including: - Binding assays used (surface plasmon resonance, ELISA, etc.) - Cell-based functional assays - In vivo mechanistic studies - Biochemical and biophysical techniques - Controls and validation methods - Any limitations of the experimental approaches noted - **Key Mechanistic Findings**: Extract the main conclusions about aflibercept’s mechanism as a soluble decoy receptor that binds VEGF-A and PlGF, including: - Primary mechanistic insights discovered - Novel aspects of the decoy receptor function - Clinical relevance of the mechanistic properties - Any unexpected or surprising mechanistic findings - Implications for therapeutic efficacy based on mechanism - Outstanding mechanistic questions identified # Results ## Characteristics of Included Studies Study Year Full Text Retrieved? Study Type Primary Focus Binding and neutralization of 2012 Yes In vitro binding study Binding kinetics and biological activity comparison of aflibercept, ranibizumab, and bevacizumab N. Papadopoulos et al. 2012 Yes In vitro binding study Binding kinetics and biological activity comparison of aflibercept, ranibizumab, and bevacizumab H. Deissler et al. 2014 No In vitro cell-based study Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal endothelial cells Atsuhiro Kanda et al. 2015 Yes In vitro and ex vivo study Aflibercept trapping of galectin-1 in diabetic retinopathy D. MacDonald et al. 2016 Yes In vitro biochemical study VEGF binding stoichiometry and immune complex formation comparison between aflibercept and bevacizumab T. Torimura et al. 2016 No In vitro and in vivo mouse model Antiangiogenic and antitumor activities in hepatocellular carcinoma F. Lazzara et al. 2019 No In vitro and in vivo diabetic retinopathy model Aflibercept regulation of retinal inflammation via PlGF/ERK pathway T. Torimura et al. 2011 No In vitro and in vivo mouse hepatoma model Antiangiogenic mechanisms in hepatoma P. Golik & K. Tońska 2012 No Review/comparison Biological principles comparison of monoclonal antibody vs decoy receptor approaches Hoai-Thu Thai et al. 2011 Yes Phase I pharmacokinetic study Mechanism-based pharmacokinetic modeling of free and bound aflibercept toof Pageof The included studies employed diverse experimental approaches to characterize aflibercept’s mechanism, with five full-text articles and five abstract-only sources. Studies spanned from 2011 to 2019, with primary methodologies including in vitro binding assays, cell-based functional assays, in vivo animal models, and pharmacokinetic modeling. ## Binding Properties ### Affinity and Kinetics Aflibercept demonstrated subpicomolar affinity for all isoforms of human VEGF-A tested, with Kd values of 0.490 pM for VEGF-A165 and 0.5 pM in pharmacokinetic studies. For PlGF-2, aflibercept exhibited Kd values of 38.9 pM for human PlGF-2 and 3.32 pM for murine PlGF-2. The association rate for aflibercept binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. ### Stoichiometry and Complex Formation A critical distinction of aflibercept’s mechanism is its formation of homogeneous 1:1 complexes with each VEGF dimer. This stoichiometry was consistent across different molar ratios and contrasted sharply with bevacizumab, which formed large multimeric complexes with dimeric VEGF165. The 1:1 binding ratio was maintained for both VEGF-A and PlGF. ### Species Specificity Aflibercept bound VEGF-A with high affinity across multiple species, including human, mouse, rat, and rabbit. The binding affinity for VEGF-A was consistent across these species, and aflibercept also bound both human and murine PlGF-2. ## Structural Mechanism ### Domain Architecture Aflibercept functions as a soluble decoy receptor composed of the second Ig domain of human VEGFR1 and the third Ig domain of human VEGFR2, fused to the Fc region of human IgG1. The VEGFR1 and VEGFR2 domains are responsible for ligand recognition and binding, while the Fc portion does not participate in target molecule recognition. ### Binding Sites and Molecular Interactions Aflibercept’s unique binding mechanism not only blocks the amino acids necessary for VEGFR1/R2 binding but also occludes the heparin-binding site on VEGF165. This dual blockade represents a distinct binding mode compared to antibody-based approaches. Aflibercept bound both heparin-binding and non-heparin-binding isoforms of VEGF-A, indicating that heparin-binding sites were not required for aflibercept’s interaction with VEGF-A. ## Decoy Receptor Function ### Prevention of Native Receptor Activation Aflibercept prevented VEGF-A and PlGF from binding to their native receptors, VEGFR1 and VEGFR2. The high-affinity binding of aflibercept to these ligands effectively sequestered them from circulation, reducing their availability for endogenous receptors. ### Inhibition of Receptor Phosphorylation Aflibercept suppressed phosphorylation of VEGFR1 and VEGFR2. In cell-based bioassays, aflibercept inhibited activation of VEGFR1 and VEGFR2, as well as VEGF-A-induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only aflibercept bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. ### Mechanism of Action The mechanism likely involved competitive inhibition, as aflibercept directly competed with native receptors for binding to VEGF-A and PlGF. The high affinity of aflibercept for these ligands suggested a strong competitive inhibition mechanism, effectively trapping these ligands and preventing their interaction with native receptors. The formation of monomeric 1:1 complexes distinguished aflibercept from bevacizumab’s multimeric complexes and represented a non-competitive inhibition mechanism that did not rely on multimeric complex formation. ## Downstream Signaling Effects ### Impact on Signaling Pathways Aflibercept suppressed phosphorylation of PI3K, AKT, and mTOR pathways. In retinal endothelial cells, both aflibercept and anti-PlGF antibody exerted protective effects by inhibiting the ERK pathway. Aflibercept eliminated VEGFR2 phosphorylation up-regulation caused by galectin-1, with downstream effects involving phospholipase C-γ and MAPK cascades leading to cell proliferation and angiogenesis. ### Cellular Response Modulation Aflibercept reduced endothelial cell proliferation, migration, and permeability. It specifically inhibited proliferation of retinal endothelial cells stimulated with VEGF-A, PlGF, or combinations of these factors. Treatment with aflibercept at therapeutically achievable concentrations reduced VEGF-A-stimulated endothelial cell migration not only to normal values but below basal level. The VEGF-A-disturbed barrier was completely restored by treatment with ≤25 μg/ml aflibercept. ### Effects on Angiogenic Processes Aflibercept suppressed differentiation of bone marrow cells to endothelial progenitor cells (EPCs) and their migration to tumor tissues. In mouse hepatoma models, aflibercept reduced microvessel density and inhibited endothelial cell proliferation and EPC differentiation. The suppression of VEGF expression in tumor tissue by aflibercept administration indicated a feedback effect on VEGF expression. ## Ligand Specificity Profile Ligand Binding Status Relative Affinity Species VEGF-A (all isoforms) Binds Subpicomolar (Kd = 0.49-0.5 pM) Human, mouse, rat, rabbit PlGF Binds Kd = 38.9 pM (human), 3.32 pM (murine) Human, murine VEGF-B Binds Kd = 3.32 pM Not specified VEGF-C Does not bind N/A Human VEGF-D Does not bind N/A Human toof Pageof Aflibercept’s broader specificity, recognizing and binding VEGF-A, VEGF-B, and PlGF, followed the specificity of VEGFR-1 and VEGFR-2. A novel finding was aflibercept’s ability to bind galectin-1 with higher affinity than VEGFR1-Fc and VEGFR2-Fc, an interaction abolished by deglycosylation of aflibercept with peptide:N-glycosidase F. This cross-reactivity with galectin-1 represented an unexpected mechanistic property beyond the VEGF family. ## Mechanistic Comparison to Other Anti-VEGF Agents Property Aflibercept Ranibizumab Bevacizumab Molecular type Soluble decoy receptor Monoclonal antibody fragment Monoclonal antibody VEGF-A binding Subpicomolar affinity, fast association Lower affinity Lower affinity PlGF binding Yes No No VEGF-B binding Yes No No Complex formation 1:1 with VEGF dimers Not specified Multimeric complexes Fc fragment effects Does not increase affinity for Fcγ receptors N/A Increased binding to Fc receptors in multimeric form Heparin-binding site Occludes on VEGF165 Not mentioned Does not occlude toof Pageof The soluble decoy receptor mechanism of aflibercept provided advantages in terms of binding affinity and potency compared to antibody-based mechanisms. Unlike bevacizumab:VEGF multimeric complexes, the monomeric aflibercept:VEGF complex did not exhibit increased affinity for low-affinity Fcγ receptors, did not activate platelets, nor did it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept. Aflibercept’s mechanism avoided avidity-driven Fc to Fcγ receptor interactions seen with bevacizumab. ## Synthesis The findings across studies were remarkably consistent regarding aflibercept’s core mechanistic properties. All studies that examined binding confirmed subpicomolar to low picomolar affinity for VEGF-A and PlGF, with the precise Kd values varying slightly based on methodology (SPR vs solution binding). The 1:1 stoichiometry was uniformly reported across biochemical studies, distinguishing aflibercept from antibody-based approaches. A key mechanistic distinction emerged in how aflibercept’s decoy receptor function differed from antibody mechanisms. Studies using in vitro binding assays demonstrated that aflibercept’s VEGFR1/VEGFR2 domains enabled broader ligand recognition (VEGF-A, VEGF-B, PlGF) compared to antibodies targeting only VEGF-A. This broader specificity was considered beneficial in contexts where multiple VEGF family members contribute to pathology. The formation of monomeric 1:1 complexes rather than multimeric structures provided safety advantages by avoiding platelet activation and immune complex-mediated effects observed with bevacizumab. The downstream signaling effects showed consistent patterns across different cell types and disease models. Studies in retinal endothelial cells, hepatoma models, and human retinal microvascular endothelial cells all demonstrated that aflibercept prevented receptor phosphorylation and downstream pathway activation. The specific pathways affected (PI3K/AKT/mTOR, ERK, MAPK) varied based on cell type and experimental conditions, but all pointed to effective blockade of angiogenic signaling. One unexpected finding was aflibercept’s interaction with galectin-1, an angiogenic factor independent of VEGF-A. This glycosylation-dependent binding represented a novel anti-angiogenic function beyond VEGF family antagonism. The clinical relevance of this interaction remained unclear, as bevacizumab pretreatment reduced VEGF-A but not galectin-1 levels in proliferative diabetic retinopathy eyes, suggesting galectin-1 may contribute to anti-VEGF resistance. The concentration-dependent effects of aflibercept revealed important considerations for therapeutic dosing. At therapeutically achievable concentrations (≤25 μg/ml), aflibercept specifically restored VEGF-A-disturbed barrier function without affecting normal barrier function. However, at high concentrations, aflibercept interfered with normal retinal endothelial cell function, suggesting that dose reduction might be considered to minimize potential adverse effects while maintaining therapeutic efficacy. This concentration-dependent specificity distinguished aflibercept from non-specific effects observed with high concentrations of IgG-containing proteins. ## References [T. Torimura, T. Ueno, H. Iwamoto, Toru Nakamura, H. Koga, and 2 more\\ (2011).Abstract 4248: Antiangiogenic mechanisms of Aflibercept in mouse hepatoma model. Cancer Research](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-58557739/index.html) [No authors found (2012).Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-9049238/index.html) [N. Papadopoulos, Joel Martin, Q. Ruan, A. Rafique, M. Rosconi, and 5 more\\ (2012).Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-254185089/index.html) [H. Deissler, G. Lang, G. Lang\\ (2014).Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal microvascular endothelial cells. Experimental Eye Research](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-27734375/index.html) [F. Lazzara, A. Fidilio, Chiara Bianca Maria Platania, G. Giurdanella, S. Salomone, and 7 more\\ (2019).A flibercept regulates retinal inflammation elicited by high glucose via the PlGF/ERK pathway. Biochemical Pharmacology](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-198966122/index.html) [P. Golik, K. Tońska\\ (2012).\[Comparison of the biological principles underlying the action of monoclonal antibody (mAb) and decoy receptor anti-VEGF agents--on the example of ranibizumab (anti-VEGF-A mAb) and aflibercept (decoy VEGFR1-2 receptor)\]. Klinika oczna](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-3011780/index.html) [Hoai-Thu Thai, C. Veyrat-Follet, N. Vivier, C. Dubruc, G. Sanderink, and 2 more\\ (2011).A mechanism-based model for the population pharmacokinetics of free and bound aflibercept in healthy subjects. British Journal of Clinical Pharmacology](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-13722650/index.html) [Atsuhiro Kanda, K. Noda, W. Saito, S. Ishida\\ (2015).Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic Retinopathy. Scientific Reports](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-205683970/index.html) [D. MacDonald, Joel Martin, Kathirselvam Muthusamy, Jiann‐Kae Luo, Erica A. Pyles, and 17 more\\ (2016).Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes. Angiogenesis](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-254186230/index.html) [T. Torimura, H. Iwamoto, Toru Nakamura, Mitsuhiko Abe, Yu Ikezono, and 7 more\\ (2016).Antiangiogenic and Antitumor Activities of Aflibercept, a Soluble VEGF Receptor-1 and -2, in a Mouse Model of Hepatocellular Carcinoma. Neoplasia](/content/review/5aaba73d-c31a-4dc6-bcb4-c2ac68655258/source/ss-18855594/index.html) Download BIBDownload RISDownload TXT ## Report Status Gather sources 200 sources found Details Screen sources 10 sources included Details Extract data 80 data points extracted Details Generate report Save PDF BIBLaTeX, ZoteroRISZotero, MendeleyTXTAPA BibliographyPDFPDFDOCXMicrosoft Word Chat Got some follow-up questions? 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Papadopoulos Angiogenesis· 2016· 0 citations SourceDOI Plain textPDF Searching for PDF Unable to find PDF from source Back Binding Properties \- Binding stoichiometry: Aflibercept forms a homogenous 1:1 complex with each VEGF dimer. \- Binding affinity and kinetics: Not mentioned. \- Comparison of binding strength to VEGF-A vs PlGF: Not mentioned. \- Differences between VEGF-A isoforms: Not mentioned. \- Temperature, pH, or other conditions affecting binding: Not mentioned. Structural Mechanism \- Domains involved: Second Ig domain of human VEGFR1 and third Ig domain of human VEGFR2. \- Specific amino acid residues or regions: Not specified. \- Sites blocked on VEGF-A/PlGF: Amino acids necessary for VEGFR1/R2 binding and heparin-binding site on VEGF165. \- Heparin-binding sites: Occluded. \- Complex formation: 1:1 complex with VEGF dimers and PlGF. \- Structural differences in binding to VEGF-A vs PlGF: Similar stoichiometry for both. Decoy Receptor Function Aflibercept functions as a decoy receptor by binding VEGF-A and PlGF, preventing them from interacting with native VEGFR1 and VEGFR2. It blocks the amino acids necessary for receptor binding and occludes the heparin-binding site on VEGF165. Aflibercept forms a 1:1 complex with VEGF dimers, sequestering them and reducing their availability for endogenous receptors. This mechanism is non-competitive, as it does not rely on multimeric complex formation or increased binding to low-affinity Fc receptors. Downstream Signaling Effects \- Effects on VEGFR1/VEGFR2 phosphorylation and activation: Aflibercept blocks the amino acids necessary for VEGFR1/R2 binding. \- Impact on downstream pathways: Minimal impact due to lack of avidity-driven Fc to Fcc receptor interactions. \- Changes in cellular responses: Minimal effects on proliferation, migration, and permeability due to lack of platelet activation. \- Effects on angiogenic processes: Reduction in angiogenic processes due to blockade of VEGF-A and PlGF pathways. \- Feedback effects on VEGF/PlGF expression: Not explicitly mentioned. \- Comparison of signaling blockade for VEGF-A vs PlGF pathways: Aflibercept binds both, suggesting a broad blockade of angiogenic signaling. Ligand Specificity Profile \- Aflibercept binds: VEGF-A, PlGF, VEGF-B \- Aflibercept does not bind: Not mentioned \- Relative binding strengths: Not mentioned \- Species specificity: Human \- Cross-reactivity: Not mentioned Mechanistic Comparison \- Aflibercept is a soluble decoy receptor that forms a 1:1 complex with VEGF dimers, distinct from the multimeric complexes formed by bevacizumab. \- Aflibercept's mechanism involves blocking VEGFR1/R2 binding sites and occluding the heparin-binding site on VEGF165, which is unique compared to antibody-based approaches. \- The 1:1 complex formation does not increase affinity for low-affinity Fcγ receptors or activate platelets, unlike multimeric complexes. \- Aflibercept's mechanism avoids avidity-driven Fc to Fcc receptor interactions seen with bevacizumab. \- The soluble decoy receptor mechanism is advantageous in avoiding multimeric complex formation and its associated effects. Experimental Methods \- Binding assays: Surface plasmon resonance (SPR) to determine dissociation rate constants to Fcc receptors. \- Cell-based functional assays: Platelet activation using light aggregometry and serotonin release models. \- In vivo mechanistic studies: Use of FccRIIa transgenic mice to assess thrombocytopenia and thrombosis. \- Biochemical and biophysical techniques: Multiple-angled laser light scattering (MALLS) coupled to size exclusion chromatography (SEC) for binding stoichiometry. \- Controls and validation methods: Use of human Fc as a control. \- Limitations: Not explicitly mentioned. Key Mechanistic Findings \- Primary mechanistic insights discovered: Aflibercept is a novel soluble decoy receptor that binds VEGF-A and PlGF, forming a homogenous 1:1 complex with VEGF dimers. \- Novel aspects of the decoy receptor function: Unique binding stoichiometry and occlusion of the heparin-binding site on VEGF165. \- Clinical relevance of the mechanistic properties: Safer profile with less platelet activation and cell surface binding compared to bevacizumab. \- Unexpected or surprising mechanistic findings: Occlusion of the heparin-binding site on VEGF165. \- Implications for therapeutic efficacy based on mechanism: Unique binding mechanism may contribute to effectiveness in treating wet AMD with fewer adverse effects. \- Outstanding mechanistic questions identified: Long-term efficacy and safety implications of the unique binding mechanism in various clinical settings. Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165. Introduction Angiogenesis, the growth of new blood vessels from preexisting vasculature, is a highly orchestrated process that is critical for proper embryonic and postnatal vascular development \[1\]. Abnormal or pathological angiogenesis is a hallmark of cancer and several retinal diseases where the upregulation of proangiogenic factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), leads to increases in endothelial proliferation, changes in vasculature morphology, and increased vascular permeability \[2,3\]. In particular, blockade of VEGF has shown clinical utility in the oncology setting as well as several retinal vascular diseases characterized by abnormal angiogenesis and/or vascular permeability, such as the ''wet'' form of age-related macular degeneration (AMD), the leading cause of blindness in the elderly \[4,5\]. The formation of new blood vessels and vascular leakage in wet AMD leads to macular edema, thickening of the retina, and loss of vision. Elevated levels of VEGF have been found in the vitreous fluid and retinal vasculature of patients with AMD \[6\]. Blocking VEGF activity has also become the therapy of choice for treating diabetic macular edema (DME), retinal vein occlusions, and other ocular diseases where abnormal angiogenesis is the underlying etiology \[7\]\[8\]\[9\]\[10\]. Several anti-VEGF therapies have been approved for use in neovascular or ''wet'' AMD. Pegaptanib, an oligonucleotide aptamer that binds the heparin-binding domain of VEGF 165 , was the first anti-VEGF therapy that showed some benefit in treating wet AMD, although most patients still experienced visual decline \[11\]. The introduction of ranibizumab, an affinity-matured, humanized monoclonal antibody fragment (Fab), represented a major advance in treating wet AMD patients since it was not only able to stabilize but in many cases improve visual acuity \[12\]. However, prior to regulatory approval of ranibizumab, ophthalmologists began using bevacizumab, a humanized monoclonal antibody to VEGF already approved for metastatic colorectal cancer, to treat wet AMD. Like ranibizumab, bevacizumab binds to all isoforms of VEGF-A, but is aliquoted and repackaged for offlabel use in ophthalmological indications \[13\]. Several, small, non-randomized studies evaluated bevacizumab as a potential treatment option in the treatment for wet AMD and thus began the ongoing debate on the merits of utilizing bevacizumab for the treatment for diseases affecting the ocular vasculature \[14\]. More recently, in a large (n = 1208), prospective, randomized comparison of agerelated macular degeneration treatments trial (CATT), comparison of the same regimens of ranibizumab or bevacizumab demonstrated similar improvements for the primary end point of visual acuity at 1 year \[15\]. However, somewhat higher rates of systemic serious adverse events (SAEs) were observed in patients treated with bevacizumab compared to ranibizumab. Curiously, the excess in systemic SAEs observed in bevacizumab-treated patients in the CATT trial did not correspond to adverse cardiovascular events, such as hypertension and arteriothrombolic events (ATEs), previously known to be caused by systemic VEGF inhibition in oncology trials that employed much higher, intravenous doses of bevacizumab. Rather, patients treated intravitreally with low-dose bevacizumab exhibited increases in SAEs affecting other organ systems, particularly gastrointestinal disorders. While two additional, but smaller trials (IVAN; n = 610, MANTA; n = 321) individually showed no statistically significant differences in systemic SAEs between ranibizumab and bevacizumab \[16,17\], a recent meta-analysis (n = 3665) comprising the above studies as well as six additional trials also found a higher incidence of gastrointestinal disorders in patients treated with intravitreal bevacizumab compared to ranibizumab, with no differences in other systemic SAEs \[18\]. In comparing the safety profile of ranibizumab to bevacizumab when treating DME, the recent Protocol T trial showed no differences in the rates of serious adverse events among all three anti-VEGF treatments (aflibercept, bevacizumab, ranibizumab), though visual acuity gains with aflibercept were significantly greater than with ranibizumab or bevacizumab in the overall population, and these differences were especially evident in patients with poor vision at the start of treatment \[19\]. Although mechanisms underlying the potentially higher rate of systemic SAEs not typically associated with anti-VEGF activity in patients receiving intravitreal bevacizumab remains unknown, a number of investigators have pointed out several fundamental differences between ranibizumab and bevacizumab. First, ranibizumab is a Fab fragment and is significantly smaller in size than a fulllength antibody. This was advantageous for the development for intravitreal injection as the smaller size of ranibizumab was expected to enhance diffusion from the vitreous into the retina and choroid \[20\]. VEGF and closely related molecules are ligands for a family of related receptor tyrosine kinases (VEGFRs), and as such exist naturally as dimers. Since ranibizumab is a Fab fragment, two molecules of ranibizumab are bound by each VEGF dimer. In contrast, bevacizumab is an IgG comprising two Fabs as well as an Fc domain. Due to its bivalent nature, a single bevacizumab molecule can bind both active sites in a single VEGF dimer. Interestingly, it has also been reported that bevacizumab has the capacity to form large multimeric complexes with VEGF \[21,22\]. Second, though derived from bevacizumab, ranibizumab has been affinity-matured with a reported affinity enhancement of 10-to 100-fold relative to the Fab, which is associated with a similarly enhanced activity of ranibizumab in cell-based assays, compared to the Fab fragment of bevacizumab \[23,24\]. However, the bivalent nature of the full-length bevacizumab antibody contributes substantially to its ability to bind and neutralize VEGF. Specifically, avidity interactions of bevacizumab with its dimeric VEGF target significantly increase binding affinity and potency of the bivalent antibody relative to the monovalent antigenbinding fragment \[25\]. Lastly, ranibizumab does not contain a fragment crystallizable (Fc) region, unlike full-length antibodies. While the Fc moiety is useful to increase circulatory half-life when an antibody is given systemically, via its interactions with the neonatal receptor, the Fc region can also promote effector function if the antibody is multimerized upon binding to its target. In the absence of this multimerization, the Fc domain of therapeutic antibodies rarely occupies Fcc receptors due to the constant competition with high levels of endogenous IgG (5-15 g/L) \[26\]. Meyer et al. \[22\] have shown that bevacizumab and equimolar amounts of VEGF 165 can form immune-like complexes, which in the presence of heparin can engage the FccRIIa receptor on thrombocytes leading to platelet activation. Other studies have also reported differences in the activities of ranibizumab and bevacizumab that appear to be unrelated to VEGF neutralization per se. For example, bevacizumab exhibits enhanced binding to and accumulation in retinal pigment epithelium (RPE) cells in vitro, relative to ranibizumab \[27\]. Aflibercept is a novel soluble decoy receptor consisting of an all-human amino acid sequence comprising the second Ig domain of human VEGFR1 and the third Ig domain of human VEGFR2 expressed as an inline fusion with the constant region (Fc) of human IgG1 \[28\]. Like bevacizumab and ranibizumab, aflibercept binds all forms of VEGF-A (VEGF) but in addition binds PlGF and VEGF-B \[25\]. Aflibercept was recently approved for the treatment for wet AMD based on the VIEW studies \[29\]. These trials demonstrated equivalent improvements in visual acuity at 1 year between patients treated monthly with ranibizumab and those treated with aflibercept every other month following three initial monthly loading doses. Each aflibercept molecule, like bevacizumab, contains two, independent VEGF binding arms held together via the Fc moiety. Upon binding VEGF dimers, aflibercept could in theory form large, multimeric immune-like complexes similar to those previously described for bevacizumab, which in turn could trigger processes mediated by low-affinity Fc receptors. In the present study, we compare the binding stoichiometries of bevacizumab and aflibercept to VEGF 165 , over a range of relative molar concentrations, and also assessed the binding affinities of bevacizumab and aflibercept to VEGF 121 , VEGF 165 , and Fcc receptors, alone or when complexed with VEGF. The ability of bevacizumab:VEGF and aflibercept:VEGF complexes to activate human platelets in vitro, and the propensity of both bevacizumab and aflibercept to bind to cultured human retinal epithelial (ARPE-19) and human umbilical vein endothelial cells (HUVEC) in the presence and absence of VEGF 165 and VEGF 121 also were assessed. The results of these experiments confirm that bevacizumab can form large multimeric complexes with VEGF, when both antibody and ligand are present in roughly (within tenfold) equimolar concentrations. In contrast, aflibercept forms a homogenous 1:1 complex with VEGFs over all molar ratios tested. Moreover, in contrast to the large and heterogenous bevacizumab:VEGF 165 complexes, these discrete aflibercept:VEGF 165 complexes do not activate platelets in the presence of heparin. Finally, aflibercept does not bind to the surface of HUVEC or ARPE-19 cells to an appreciably greater degree than the control, human Fc (hFc), in the presence or absence of VEGFs. In contrast, binding of bevacizumab to the surface of these cells was greatly enhanced in the presence of exogenous or endogenous VEGF 165 , but not VEGF 121 . This finding suggested that the cell surface binding of bevacizumab:VEGF 165 complexes was mediated by the heparin and/or neuropilin-1 (NRP1)binding domains of this VEGF isoform. This was confirmed by subsequent surface plasmon resonance (Biacore) experiments, which showed that bevacizumab:VEGF 165 complexes, but not aflibercept:VEGF 165 complexes, were readily bound by surface-captured NRP1 or heparin. In conclusion, under certain conditions, bevacizumab can form large, multimeric immune-like complexes that exhibit enhanced binding not only to a range of Fcc receptors, but also, when complexed with VEGF 165 , to heparin and neuropilin present on the surfaces of cells. In contrast, aflibercept exclusively forms a 1:1 complex with VEGF dimers, which does not appreciably increase binding to low-affinity Fc receptors, heparin, or neuropilin-compared to unbound aflibercept or control, monomeric IgGs. The fundamental difference between bevacizumab and aflibercept behavior appears to be attributable to the unique way in which the aflibercept encompasses or ''Traps'' a single, VEGF dimer, not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF as well. Results In contrast to bevacizumab, aflibercept forms a 1:1 complex with VEGF Multiple-angled laser light scattering (MALLS) coupled to size exclusion chromatography (SEC) was used to calculate the stoichiometry of bevacizumab:VEGF 165 and aflibercept:VEGF 165 complexes. VEGF 165 was mixed with either bevacizumab or aflibercept in solution at molar ratios of inhibitor to ligand of between 5:1 and 1:5. Bevacizumab was found to form a heterogeneous mixture of multimeric complexes in the presence of VEGF 165 , with molar masses ranging from \*330 to 700 kDa (Fig. 1a ). In contrast, aflibercept at a 1:5 molar ratio showed two discrete peaks (Fig. 1b ), as expected based on previous results \[21\]. The earlier eluting peak, with an apparent molar mass of 157 kDa, corresponded to a 1:1 complex between aflibercept (\*115 kDa) and VEGF 165 (\*40 kDa). The latter eluting peak corresponded to the expected excess of free VEGF 165 dimer (based on the average molar mass of the peak and comparison with the elution times from VEGF 165 injections). Analysis of a preformed aflibercept:VEGF 165 complex at a 5:1 molar ratio showed a single peak of 119 kDa (Fig. 1b ), which is attributed to incomplete separation between the 1:1 aflibercept:VEGF 165 complex (\*157 kDa) and the excess free aflibercept (\*115 kDa). SEC-MALLS analysis was also used to estimate the binding stoichiometry and molar mass of the complexes formed between bevacizumab or aflibercept and PlGF-2 (Figure S1 ). No complex formation was observed between bevacizumab and PlGF-2 (Figure S1A ). The aflibercept:PlGF-2 complex demonstrated a very similar stoichiometry to the aflibercept:VEGF 165 complex, with a single major homogenous peak (molar mass of 150 kDa) corresponding to a 1:1 complex between aflibercept and PlGF-2 (\*42 kDa) and a minor peak corresponding to an excess of free PlGF-2 dimer (Figure S1B ). Aflibercept's binding half-life to Fcc receptors does not change in the presence of VEGF Surface plasmon resonance was used to determine the dissociation rate constants (k d ) of bevacizumab and aflibercept to the family of human Fcc receptors in the presence or absence of VEGF 165 or VEGF 121 (Table 1 ). Equilibrium binding constants (K D = k d /k a ) were not calculated since the heterogenous nature of the bevacizumab:VEGF complex did not allow accurate molar concentrations to be utilized and thus association rate constants (k a ), which are dependent on concentration, could not be determined. Bevacizumab and aflibercept, in the absence of VEGF, exhibited the expected rapid dissociation rate constant (k d ) from low-affinity Fc receptors, with \[30\]. However, when bevacizumab was pre-incubated in a 1:1 molar ratio with VEGF 165 or VEGF 121 , the resulting complexes exhibited longer t 1/2 values (slower dissociation rate constants) when bound to the low-affinity Fcc receptors FccRIIa, FccRIIb, FccRIIIa (176F), FccRIIIa (176 V), and FccRIIIb compared to bevacizumab alone. This increase in binding half-life ranged from a ninefold increase when the bevacizumab:VEGF complex was flowed over FccRIIIa (176 V) to a 177-fold increase in t 1/2 when flowed over FccRIIb. In contrast, aflibercept with or without VEGF present exhibited no change in t 1/2 values when bound to Fcc receptors. Representative sensorgrams of bevacizumab and aflibercept binding to Fcc receptors are shown in Figures S2, S3 , S4. Aflibercept:VEGF 165 complexes do not activate platelets Previous studies have demonstrated that, in the presence of heparin, bevacizumab:VEGF 165 complexes can activate human platelets via FccRIIa \[22\]. The potential for aflibercept-VEGF 165 complexes to induce FccRIIa-dependent platelet activation was tested using light aggregometry. Preformed complexes of aflibercept and VEGF 165 at equal molar ratios in the presence of heparin failed to induce aggregation of human platelets (Fig. 2a ). In contrast, preformed complexes of bevacizumab and VEGF 165 at equal molar ratios in the presence of heparin caused marked aggregation of platelets over a concentration range of 100-400 nM (Fig. 2b ). However, increasing the molar excess of bevacizumab (Cfourfold relative to VEGF 165 ) results in a loss of platelet activation (data not shown) Bevacizumab:VEGF complexes below 100 nM did not activate platelets. Platelet activation was not detected at concentrations below 100 nM of bevacizumab:VEGF 165 complex. Platelet activation was also tested using a serotonin release assay. The combination of aflibercept and VEGF 165 at equal molar ratios (500 nM each) in the presence of heparin was unable to stimulate serotonin release from platelets (Fig. 2c ). However, the presence of bevacizumab and VEGF 165 at an equal molar ratio of 500 nM each in the presence of heparin induced up to 80 % release of serotonin from platelets (Fig. 2d ), consistent with findings from the light aggregometry assays. Aflibercept:VEGF 165 complexes do not induce thrombocytopenia or thrombosis in FccRIIa transgenic mice Injection of preformed (1:1 molar) bevacizumab:VEGF 165 complexes along with unfractionated heparin into transgenic mice expressing human FccRIIa has been reported to cause severe thrombocytopenia and occlusive thrombosis in alveolar capillaries \[22\]. We sought to determine whether preformed aflibercept:VEGF 165 complexes in the presence of unfractionated heparin could trigger a similar set of sequelae in human FccRIIa transgenic mice. Animals receiving aflibercept:VEGF 165 complexes (1:1 molar ratio) did not exhibit these symptoms (n = 10). Platelet counts 10 min following immune complex injection demonstrated thrombocytopenia in animals receiving bevacizumab:VEGF 165 but not aflibercept:VEGF 165 complexes (Fig. 3a ). Specifically, mice injected with phosphate-buffered saline (PBS; n = 5) had baseline platelet counts of (1173 ± 179; mean ± SD), whereas animals receiving bevacizumab ? VEGF 165 had mean platelet counts of 331 ± 217 (with heparin; P \ 0.001) and 725 ± 303 (without heparin; P \ 0.003). Mice receiving aflibercept ? VEGF 165 had mean platelet counts of 966 ± 168 (with heparin) and 878 ± 250 (without heparin), which were not statistically different from baseline (PBS) animal counts. Mouse antihuman CD-40 ligand immune complexes \[22\] served as a positive control and induced severe thrombocytopenia in FccRIIa transgenic mice with mean platelet counts of 163 ± 137 (P \ 0.001). Consistent with the thrombocytopenia, mice injected with bevacizumab:VEGF 165 complexes developed abundant occlusive thrombi in the pulmonary vasculature (Fig. 3c ). In contrast, mice injected with aflibercept:VEGF 165 complexes failed to develop microvascular thrombosis (Fig. 3d ). Aflibercept exhibits no significant cell surface binding to ARPE-19 cells or HUVEC Cell surface binding of aflibercept and bevacizumab alone or in complex with VEGF was tested using ARPE-19 (Fig. 4a ) and HUVEC (Fig. 4b ). Binding studies were conducted for both inhibitors with no ligand present or following pre-incubation with VEGF 121 or VEGF 165 . Binding to ARPE-19 cells was not observed for bevacizumab or aflibercept in complex with VEGF 121 or in the absence of ligand (Fig. 4a , row 1, 3), but significant ARPE-19 cell surface binding was observed for bevacizumab:VEGF 165 complexes. Binding was not observed for the aflibercept:VEGF 165 complex (Fig. 4a , row 2). Similarly, binding to HUVEC was not observed for aflibercept or bevacizumab in complex with VEGF 121 or in the absence of ligand (Fig. 4b , row 1, 3). However, significant HUVEC cell surface binding was observed for bevacizumab:VEGF 165 complexes, and only very low levels of surface binding were observed for aflibercept:VEGF 165 complexes (Fig. 4B , row 2). Complexes binding to the cell surface of both ARPE-19 and HUVEC were found to be dependent on the molar ratio of the inhibitor:VEGF 165 complex mixtures (Figure S5 and Figure S6 ). Complexes binding to the cell surface of ARPE-19 were observed at bevacizumab:VEGF 165 ratios between 0.06:1 and 4.5:1, with the greatest binding seen at a molar ratio of 0.1:1, and complex binding to HUVEC was observed at bevacizumab:VEGF 165 ratios between 0.5:1 and 13.5:1, with maximal binding observed at a 1.5:1 molar ratio. Cell surface binding of bevacizumab:VEGF 165 complexes is dependent on heparin in ARPE-19 cells and neuropilin-1 in HUVEC Cell surface binding of bevacizumab:VEGF 165 complexes was tested in the presence of heparin or recombinant human neuropilin-1 (rhNRP-1). Pre-binding of VEGF 165 with soluble heparin concentrations of 250 nM or higher prevented binding of bevacizumab:VEGF 165 complexes to ARPE-19 cells (Fig. 5a ). No significant blocking of bevacizumab:VEGF 165 complex binding to ARPE-19 cells was observed with concentrations of rhNRP-1 of up to 100 nM (Fig. 5a ). In contrast, pre-binding of VEGF 165 with rhNRP-1 (25 nM or higher) prevented binding of bevacizumab:VEGF 165 complexes to HUVEC (Fig. 5b ), while no significant blocking of bevacizumab:VEGF 165 complex binding to HUVEC was observed in the presence of up to 1000 nM soluble heparin (Fig. 5b ). These results are consistent with surface plasmon resonance analysis of bevacizumab:VEGF 165 and aflibercept:VEGF 165 in the presence of rhNRP-1 and heparin (Fig. 6a , b, S7A and B). Bevacizumab:VEGF 165 complexes were found to bind to both rhNRP-1-and heparin-coated surfaces at concentrations as low as 1 nM. Binding of aflibercept:VEGF 165 complexes to rhNRP-1-and heparin-coated surfaces was only detected at complex concentrations of 100 nM or greater. Cell surface binding is dependent on the concentration of endogenous VEGF 165 ARPE-19 cells were used to test cell surface binding of bevacizumab and aflibercept in the presence of VEGF endogenously produced by these cells. ARPE-19 cells have been shown to express VEGF after several days in culture \[31\]. Cells in culture were incubated with aflibercept, bevacizumab, or hFc, for 3 days starting at day 2, 4, 9, and 14, and surface binding was then examined 3 days later. Aflibercept and the hFc control showed minimal surface binding at all time points. In contrast, bevacizumab showed significant cell surface binding at the Day 9-12 and Day 14-17 time points (Fig. 7c-l and 7d-1 ). Next-generation sequencing and ELISA binding were used to determine the expression level and identity of the VEGF isoforms present in the culture medium at various time points. VEGF 165 was found to be the predominant VEGF species present (Fig. 7m, n ), with the concentration of this isoform increasing as a function of ARPE-19 time in culture. The highest concentrations of VEGF 165 were found on the Day 9 and Day 28 time points, consistent with the high level of bevacizumab cell surface binding at later time points. These results closely resemble cell surface binding studies conducted with preformed bevacizumab:VEGF 165 complexes and suggest that bevacizumab can spontaneously form multimeric complexes in the presence of endogenous VEGF 165 . Discussion Several reports have indicated that bevacizumab, in contrast to ranibizumab, can bind cell surface-bound VEGF on RPE and endothelial cells \[27,32\]. Additional studies have provided evidence that bevacizumab not only binds cellsurface-bound VEGF on RPE cells, but that this binding may trigger the complement cascade resulting in cell death \[3,33\]. Components of the complement pathway are known to play a role in AMD pathogenesis, although the mechanism has not been clearly defined. Activation of the complement cascade can result in damage to retinal cells as well as surrounding tissue \[34\], and conversely, complement components can contribute to neuronal homeostasis in the retina \[35\]. Aflibercept, like bevacizumab, contains an Fc domain; however, several reports have shown that this anti-VEGF agent does not induce changes in cell morphology, induce apoptosis, or decrease cell viability of retinal cells in culture \[36,37\]. To further understand what could be driving these observed differences in vitro, we conducted a detailed analysis of bevacizumab and aflibercept when complexed with VEGF by analyzing their stoichiometry of binding, affinity to Fcc receptors, propensity to activate platelets and ability to bind ARPE-19 and HUVEC. The results of these experiments demonstrate that in contrast to bevacizumab, which can form large multimeric complexes with VEGF, aflibercept forms a homogeneous 1:1 molar complex with VEGF and PlGF. The discrete monomeric complexes of aflibercept:VEGF do not allow avidity-driven Fc to Fcc receptor interactions as seen with the large heterogeneous complexes that bevacizumab forms with VEGF when both are present at near equal molar ratios. Furthermore, since bevacizumab, unlike aflibercept, does not block heparin binding to VEGF 165 , these multimeric bevacizumab:VEGF complexes, in the presence of heparin, can activate FccRIIa receptors triggering platelet aggregation. Lastly, this fundamental difference in binding geometry explains the observations that no significant cell binding was observed for aflibercept:VEGF complexes, while the heterogeneous bevacizumab:VEGF complexes bound to the cell surfaces of both ARPE-19 and HUVEC. This report demonstrates at molar ratios of 5:1 to 1:5, aflibercept to VEGF 165 , aflibercept was observed to form a 1:1 molar complex. In contrast, bevacizumab at the same molar ratios formed high molecular weight complexes with VEGF 165 , in agreement with earlier published work \[21\]. When the affinity of these observed high molecular weight complexes to high-and low-affinity Fcc receptors was investigated, we demonstrated that bevacizumab, when bound to either VEGF 121 or VEGF 165 at equal molar ratios (\*1 lM), had drastically increased t 1/2 values, ranging from \*threefold for the FccRI receptor to \*177-fold increase for the low-affinity FccRIIb receptor, compared to bevacizumab alone. In contrast, aflibercept in complex with VEGF 121 or VEGF 165 at equal molar ratios (\*1 lM) showed no appreciable difference in t 1/2 values relative to aflibercept alone. Through platelet activation studies, we showed that when heparin was added to bevacizumab:VEGF complexes (100-400 nM), marked aggregation of platelets was observed. Heparin, in this context, allows for optimal FccRIIa receptor clustering by bringing the bevacizumab:VEGF complexes to the platelet surface \[22\]. However, when we increased the molar excess of bevacizumab (Cfourfold relative to VEGF 165 ), the ability to trigger platelet activation was lost, most likely due to the ability of excess bevacizumab to decrease the size of the multimeric complexes, reducing the avidity between bevacizumab's Fc domain and the FccRIIa receptor. In contrast, preformed complexes of aflibercept and VEGF 165 at equal molar ratios in the presence of heparin failed to Representative sensograms of bevacizumab and aflibercept binding to NRP1.mFc and heparin at 5 nM are shown in Supplementary Figure 7 induce aggregation of human platelets. Consistent with these results, serotonin release from platelets was not observed for aflibercept:VEGF 165 complexes but was observed for bevacizumab:VEGF 165 complexes. These bevacizumab:VEGF 165 complexes could form when systemic or intravitreal levels of bevacizumab are low, approaching the concentrations of endogenous VEGF. To study the potential sequelae of bevacizumab's immune complex formation with VEGF, we incubated bevacizumab:VEGF 165 with either ARPE-19 cells or HUVEC. At equimolar ratios of bevacizumab to VEGF 165 , the complexes bound to the ARPE-19 cell surface. Indeed, there was significant staining of the preformed bevacizumab:VEGF 165 complexes at molar ratios from 4.5:1 to 0.06:1. These findings support the earlier work of Klettner et al. \[38\] which showed that bevacizumab was taken up by primary porcine RPE cells as assessed by confocal laser scanning microscopy and flow cytometry. There was no staining on the cell surface with bevacizumab complexed with VEGF 121 , indicating that the heparin-binding domain and/or the NRP1-binding site was required for immune complex deposition. Very little cell surface staining was observed on either ARPE-19 cell or HUVEC by aflibercept:VEGF 165 complex over a wide range of molar ratios. The binding of bevacizumab on the cell surface only when in complex with VEGF 165 but not VEGF 121 points to the involvement of the positively charged heparin-binding domain found on VEGF 165 . The heparin-binding domain confers diverse functions on VEGF 165 \[39\], including interaction with heparin and neuropilin present on the cell surface. Binding of the bevacizumab:VEGF 165 complex to ARPE-19 cells was blocked by the addition of heparin but not neuropilin. Conversely, binding of the bevacizumab:VEGF 165 complex to HUVEC was blocked only by the addition of neuropilin, illustrating that both heparin or neuropilin can anchor these complexes to the cell surface. To determine whether these immune complexes could form with endogenously produced VEGF, we added either bevacizumab or aflibercept to ARPE-19 cells after allowing VEGF production to proceed over various periods (2,4,9, and 14 days). After quantifying VEGF levels using both ELISA and next-generation sequencing, it was clear that bevacizumab cell surface staining correlated with the levels of VEGF produced. As in the studies using exogenous VEGF, the addition of aflibercept to ARPE-19 cells containing different levels of VEGF did not result in detectable cell surface staining. In conclusion, we have demonstrated that bevacizumab and aflibercept can exhibit strikingly different binding stoichiometries with VEGF. When the molar concentration of bevacizumab is within approximately tenfold that of VEGF, two molecules of bevacizumab can bind to each VEGF dimer via one of its two Fab arms, leaving the second Fab arm on each bevacizumab free to bind another VEGF dimer. This binding stoichiometry promotes the formation of large, multimeric complexes of bevacizumab which exhibit significantly enhanced binding affinity toward low-affinity Fcc receptors. Moreover, the heparinand neuropilin-binding sites of VEGF 165 are not occluded when bound to bevacizumab, such that heparin/neuropilinmediated cell surface binding is also enhanced. That large multimeric complexes of bevacizumab:VEGF 165 exhibit enhanced binding to both heparin/neuropilin and lowaffinity Fc receptors on cell surfaces underlies the ability of these complexes to promote platelet aggregation and activation. In contrast, each aflibercept molecule forms a homogenous 1:1 complex with each VEGF dimer at all molar ratios of aflibercept:VEGF tested. This 1:1 binding stoichiometry does not enhance binding to heparin or neuropilin on cell surfaces. In addition, 1:1 aflibercept:VEGF complexes do not bind low-affinity Fcc receptors more avidly than unbound aflibercept or monomeric IgG1, or cause platelet aggregation and activation. While there is no evidence to link the reported higher gastrointestinal disorders and the mechanism by which bevacizumab binds VEGF, we have shown that differences in binding at high molar ratios can be directly correlated to differences in Fcc receptor binding, cell surface binding, and platelet activation. Multi-angle laser light scattering detection coupled to size exclusion chromatography (SEC-MALLS) The SEC-MALLS system comprised an Agilent 1200 Series HPLC system equipped with an ultraviolet (UV) diode array detector coupled to a Wyatt Technology MiniDawn TREOS laser light scattering (LS) detector and an Optilab REX differential refractometer (RI) detector (Santa Barbara, CA). The detectors were connected in series in the following order: UV-LS-RI. LS and RI detectors were calibrated according to the instructions provided by Wyatt Technology. Complexes comprising 20 lM VEGF inhibitor (aflibercept \[Regeneron\] or bevacizumab \[Roche\]) and 100 lM VEGF 165 (Regeneron, produced in CHO cells) were prepared separately and incubated for 12 h at ambient temperature to form 1:5 molar ratio inhibitor:ligand complexes. Similarly, 5:1 inhibitor:ligand complexes were prepared with 100 lM VEGF inhibitor and 20 lM VEGF 165 . Complexes comprising PlGF-2 (Regeneron) and VEGF inhibitor (aflibercept or bevacizumab) were prepared in the same manner as the inhibitor:VEGF 165 complexes. Appropriate amounts of unbound inhibitor (20 lM) or ligand (40 lM) were also injected separately into a preequilibrated Superose 12 10/300 GL (GE Healthcare) size exclusion column. The mobile phase was 10 mM phosphate, 500 mM NaCl, pH 7.0 with a flow rate of 0.3 mL/ min. Molar masses of free ligands, free inhibitor, and inhibitor:ligand complexes were determined using ASTRA software (Wyatt Technology) as previously described \[40\]. The standard deviation calculated from bovine serum albumin (BSA) standard samples was within 2 % in different SEC-MALLS experiments. Surface plasmon resonance kinetic binding assay SPR experiments were performed on a Biacore 3000 instrument using a dextran-coated (CM4 or CM5) chip at 25 °C. The running buffer was filtered HBS-T (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.05 % polysorbate 20, pH 7.4). A capture sensor surface was prepared by covalently immobilizing anti-histidine monoclonal antibody (His capture kit, GE Healthcare), anti-mouse Fc (Mouse antibody capture kit, GE Healthcare), or neutravidin (Thermo Fisher Scientific Inc.) to the chip surface using 1-Ethyl-3-\[3-dimethylaminopropyl\]carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. Following surface activation, capturing reagents in coupling buffer (0.1 M acetate buffer, pH 4.5) were injected over the activated chip surface until the binding signals in resonance unit (RU) reached about 3500 RU for the anti-histidine monoclonal antibody surface, 1500 RU for the anti-mouse Fc polyclonal antibody, and 5000 RU for the neutravidin, respectively. The surfaces were then washed and treated with 10 mM glycine-HCl at pH 1.5 to remove uncoupled residual proteins. The various his-tagged or myc-myc His-tagged Fcc receptor family members, mFc-tagged human NRP1 (Regeneron), and biotinylated heparin (Sigma-Aldrich) were injected over the corresponding capture surfaces to a low density at approximate 200 RU in the running buffer with a concentration range between 5 and 10 lg/mL. The His-tagged FccRIIA R131 , FccRIIB/C, FccRIIIB, and FccRI purchased from R&D Systems, and myc-myc His-tagged FccRIIIA F176 , and FccRIIIA V176 were generated at Regeneron. Following the capture step, bevacizumab (Roche) or aflibercept (Regeneron) solutions with and without equimolar concentrations of VEGF 165 (Regeneron) or VEGF 121 (R&D Systems, produced in E. coli) (from 1.0 lM to 31.25 nM in a twofold serial dilutions) were individually injected over the various Fcc receptor, human NRP1.mFc, and biotin-heparin surfaces, and the real-time binding signals were recorded. The specific binding sensorgrams were obtained using a double-referencing procedure as described by Myszka et al. \[41\] by subtracting the binding signal on the blank capture surface and sample buffer run. The binding data were then processed and analyzed using Scrubber software (version 2.0, BioLogic Software), and the dissociation rate constant (k d ) and dissociation half-life t 1/2 were calculated. Washed platelet preparation Platelet concentrate was obtained from the NY Blood Center and used before expiration. Washed platelet suspensions were prepared from pooled platelets obtained from citrated phosphate dextrose blood using the method of Cazenave et al. \[42\]. Approximately 20 mL of platelet concentrate was centrifuged (2200g, 25 °C) for 12 min. The supernatant was removed, and 20 mL of modified Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO 3 , 0.42 mM NaH 2 PO 4 , 2 mM MgCl 2 , 5 mM HEPES, 5.55 mM dextrose, pH 6.2) containing 1.5 U/mL apyrase Grade VII (Sigma-Aldrich) and 0.5 lM prostacyclin I 2 (Cayman Chemical) was added. The platelets were resuspended and allowed to rest for 10 min at 37 °C. Following this, another 0.5 lM of prostacyclin I 2 was added, and the platelets were centrifuged (1900g, 25 °C) for 8 min. The supernatant was removed, and another 20 mL of modified Tyrode's buffer containing 1.5 U/mL apyrase Grade VII and 0.5 lM prostacyclin I 2 was used to resuspend the platelets. The platelets were allowed to rest for 10 min at 37 °C. After this second wash step, 0.5 lM of prostacyclin was added and the platelets were centrifuged (1900g, 25 °C) for 8 min. The supernatant was removed, and 40 mL of Tyrode's buffer containing calcium (Tyrode's buffer with 2 mM CaCl 2 , pH 7.35) was added. The platelets were resuspended and diluted to 300,000 cells/lL using the same buffer. Using this procedure, the washed platelet suspension was stable for 4 h when stored at 37 °C. Platelet aggregometry Platelet aggregation was monitored at an absorbance of 600 nm using a Beckman DU7400 spectrophotometer fitted with a homemade thermostatic stirring chamber. In typical experiments, 1.8 mL of platelets was added to 3-mL plastic cuvettes containing a stir bar designed for spectrophotometer cells (Fisher Scientific). The platelets, under constant stirring, were allowed to reach thermoequilibrium for 5 min. Then, 100 lL of PBS containing agonist (epinephrine, Sigma-Aldrich) and unfractionated heparin (Sigma-Aldrich) was added. After 1 min, data collection was begun and an additional 100 lL of PBS containing the bevacizumab:VEGF or aflibercept:VEGF complex was added. Platelet aggregation was typically monitored for 10-15 min. Plots of percent aggregation were calculated by taking the initial average absorbance of the platelets (\*1.3-1.4 AU) as 0 % aggregation and an absorbance of zero as 100 % aggregation. Serotonin release assay In vitro platelet activation was studied using a serotonin release model described by Meyer et al. \[22\]. Human platelets were loaded with 14 C-serotonin (PerkinElmer), then washed, and incubated at 22 °C with the test complex for 1 h under constant stirring. Released serotonin was counted in the supernatant and expressed as a percentage of the total serotonin loaded into the platelets. To create bevacizumab:VEGF 165 or aflibercept:VEGF 165 complexes, VEGF 165 (R&D Systems) and bevacizumab (Roche) or aflibercept (Regeneron) were coincubated in PBS at equimolar concentrations (2.5 lM) for 15 min at 22 °C. The complex was then added to 14 C-serotonin-loaded platelets in the presence of heparin (Sigma-Aldrich). Complex was added to platelets to obtain a final concentration of 0.1, 0.2, 0.5, or 1 lM and tested in the presence of a range of unfractionated heparin concentrations (0.5-500 lg/mL). Assessment of platelet-activating activity in FccRIIa transgenic mice Experimental animals B6;SJL-Tg (FccRIIa)11Mkz (FCGR2A) mice were obtained from Jackson Laboratories and bred in the Animal Care Facility at Florida Hospital, Orlando, in compliance with approved Institutional Animal Care and Use Committee (IACUC) guidelines and protocols. All experiments were performed in 8-to 15-week-old mice weighing between 16 and 20 g. All animals were genotyped and confirmed positive for the FccRIIa transgene according to Jackson Laboratories PCR Protocols. Some animals were injected with PBS (vehicle containing no immune components) in order to establish baseline platelet counts, as described below. Mouse blood sampling, platelet counting, and intravenous injection procedures For IV injections, mice were immobilized in a standard restrainer and warmed using a heat lamp for 2 min to dilate the tail vein. Immune complexes were preassembled by mixing antibody ? antigen at a 1:1 molar ratio (126 lg M90 \[Florida Hospital\] ? 45 lg CD154 \[Peprotech\], or 35 lg VEGF \[Peprotech, produced in E. coli\] ? either 126 lg bevacizumab \[Genentech\] or 92 lg aflibercept \[Regeneron\] ± heparin \[Sigma-Aldrich\] in PBS). Immune complexes were injected slowly in a 200-lL bolus into the lateral tail vein. Animals were then removed from their restraining device and placed in an open observation cage for monitoring. Ten minutes after reagent injection, mice were anesthetized by isoflurane inhalation. Approximately 500 lL of blood was collected by cardiac puncture via 27-gauge needles into syringes containing 50 lL of acid citrate dextrose (ACD). The blood was immediately placed in a polypropylene collection tube containing 50 lL of ACD, and platelet counts were measured using an automated cell counter. Platelet counts were adjusted for citrate solution volume. All animals were observed for signs of distress throughout the test procedure, specifically for rapid shallow breathing, hunched posture, and signs of decreased or dysfunctional locomotor activity. The combination of these symptoms is often referred to as a ''shock'' or ''thrombotic'' phenotype. The observation of such symptoms was recorded along with platelet counts per animal. Together, platelet counts and signs of physical distress constitute the primary data in this animal model. Histochemical analysis of lung sections Following blood draws, animals were killed by CO 2 inhalation. Mouse lungs were dissected en bloc from isofluorane-anesthetized animals not subjected to cardiac puncture. Lungs were rinsed in PBS and immediately placed in 10 % formalin-PBS for at least 24 h prior to paraffin embedding. Histopathology laboratory technicians cut some distance into the paraffin block (lung tissue) prior to initial sectioning. Cut sections (2 lm thick) were deparaffinized and rehydrated and then processed by an automated H&E staining process at the Florida Hospital main Histopathology Laboratory. Lungs sections from each group were analyzed microscopically for evidence of thrombosis by two independent observers. Also, reference lungs were harvested from baseline animals and processed identically as described above. Fields were captured from each section using a Nikon Eclipse 80i upright microscope equipped with Plan Fluor objectives (109, NA = 0.3, DIC; 209, NA = 0.5, DIC; 409, NA = 0.75, DIC; and 609, NA = 0.85, not DIC), a DC-Fi1 digital camera, and a DS-L2 image capture device. The eyepiece and camera had a collective magnification of 109, resulting in final image magnifications of 100, 200, 400, or 6009, respectively, with each objective lens. The capture device was activated by pressing a ''Capture'' button. The image capture device was set by integrated software to automatically adjust for white balance. Observers were instructed to adjust lamp intensity and stage diaphragm (aperture) settings as needed per image; thus, variations in brightness, contrast, and color hue may be observed in some images. Images were converted from TIF format to JPEG and processed in Adobe Photoshop Elements (v8) without region-specific modifications (i.e., all changes were global, and no regionspecific change was made to any image). Statistical analysis Statistical analysis was performed using SigmaPlot v11 software. The distributions of experimental data sets for mouse platelet counts were determined by normal probability analysis. All groups were found to be normally distributed and of equal variance. Therefore, differences between groups were analyzed by one-way ANOVA using the raw data (rather than the means and standard deviations). The statistical test used was the multiple comparisons versus control group Bonferroni t test (alpha = 0.050:1.000). Cell surface binding of VEGF inhibitors to ARPE-19 cells in the presence of exogenous VEGF Confluent ARPE-19 cells (ATCC) on 96-well collagencoated plates (Greiner CellCoat) were incubated with 5 nM bevacizumab (Roche), or aflibercept (Regeneron) alone or in the presence of 10 Acknowledgements Acknowledgments The authors thank Katrina Fracchiolla for assistance in preparing the manuscript and We also thank our research associates for their assistance in the conduct of these studies, and our colleagues at Regeneron, Sanofi, and Bayer HealthCare for their critical reading of this manuscript, and many helpful comments and suggestions.Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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